Circulating Tumor Cell Detection In Hepatocellular Carcinoma Based On Karyoplasmic Ratios Using Imaging Flow Cytometry

Background & Aims:At present,circulating tumor cells(circulating tumor cells,CTCs)detection method is limited to specific or accurate cell surface marker.Because the specific cell surface marker can only express in a very small part of the cell population.Therefore,through these methods,we can only detect a small population of CTCs circulating in peripherial blood.Here we present a new CTC detection technology,which is based on the detection of high nucleocytoplasmic ratio.With it,we aim to detect larger CTCs population and enhance the sensitivity of CTC detection.Methods:First,we used quantitative imaging flow sight to analysis the peripherial blood cells of hepatocellular carcinoma patients,and found a special mononuclear cells population,which was CD45-and had higher cytoplasmic ratio and the potency of cell division compared to other cells population.We deduced that it may be a population of tumor cells and defined it as high karyoplasmic ratio cells(high karyoplasmic ratios,HKR cells).Then,we analyzed the peripheral blood of 10 HCC patients with microvascular invasion(microvascular invasion,MVI)and 5 normal people for further test.Then,in order to quantify the relationship between the number of HKR cells and the presence of MVI,we recruited 12 normal cases,12 cases of non HCC patients(cirrhosis of the liver),52 cases of liver cancer patients(18 cases,18 cases of stage MVI0 MVI1 patients,16 patients with stage MVI2).To ensure the accuracy of this research,at the same time with image flow sight detection,we also perform the traditional detection method with CD45,EpCAM,DAPI markers.To evaluate the sensitivity and specificity of our method in diagnosis of hepatocellular carcinoma and presurgery prediction of the presence of MVI,we also perform the the receiver operating characteristic curve(Reciever Operation,Curves,ROC).At the same time,we made a one-year follow-up of all cancer patients in this research to determine the clinical value of our method in predicting surgery prognosis of these patients.Finally,we randomly recruited another 42 patients and 52 liver cancer patients for prospective syudy to confirm the accuracy of the method.Results:In HCC patients,a total of 56.2 ± 23.8/100,000 cells with high karyoplasmic ratios(HKR cells)were detected in cancer patients,which was much higher than that in the non-cancer group(7.6 ± 1.2/100,000).And there was also a siginificant difference in HKR cells number between liver cancer patients with and without MVI.The receiver operating characteristic curve analysis showed that the threshold is 21.8 /100000,the area under the curve is greater than the traditional detection methods(such as CD45 dependent and EpCAM labeling).These results indicate that the new CTC detection method was more sensitive and reliable than existing methods.Conclusions:In this study,based on high karyoplasmic ratio,we developed a new quantitive metheod for CTC detection in peripheral blood with image flow sight.This method relies on the detection of high karyoplasmic ratio,which is shared by all tumor cells,not a tumor specific biomarker.Compared with traditional methods based on specific biomarkers,our method can detect larger population of CTC in peripherial blood.So it is more sensitive and may be used to moniter the conditions and prognosis of cancer patients.